Dot-blot hybridization was applied in this paper to detect five viruses infecting cucurbitaceous crops, Zuccini yellow mosaic virus （ZYMV）, Watermelon mosaic virus （WMV）, Cucumber mosaic virus （CMV）, Papaya ring...Dot-blot hybridization was applied in this paper to detect five viruses infecting cucurbitaceous crops, Zuccini yellow mosaic virus （ZYMV）, Watermelon mosaic virus （WMV）, Cucumber mosaic virus （CMV）, Papaya ringspot viruswatermelon strain （PRSV-W） and Squash mosaic virus （SqMV）, as a good alternative assay in seed health test and epidemiological and transgenic research. Digoxigenin-labelled cDNA probes of the five viruses were synthesized by PCR with the specific primers and applied in dot-blot hybridization to detect five viruses in crude extraction of the infected leaves. And three SqMV probes of different lengths （0.55, 1.6, and 2.7 kb, respectively） were designed to investigate the effect of hybridization. The results showed that the sensitivity for detecting the crude extraction of infected leaves by ZYMV, WMV, CMV, PRSV-W, and SqMV was down to 1：160, 1：160, 1：320, 1：160, and 1：320, respectively. Three SqMV probes of different length showed no differences on the sensitivity and specificity. The digoxigenin-labelled probes prepared by PCR could be used for accurate and rapid identification of 5 viruses infecting cucurbitaceous crops with good stabilities, sensitivities, specificity, and reproducibilifies.展开更多
To understand the distribution of Apple stem pitting virus （ASPV） in tissues of pear tree and provide directly theorical and technical support for shoot tips detoxication, leaves and shoot tips of Korla pear were us...To understand the distribution of Apple stem pitting virus （ASPV） in tissues of pear tree and provide directly theorical and technical support for shoot tips detoxication, leaves and shoot tips of Korla pear were used as the materials, cDNA probe for ASPV was synthesized through RT-PCR reaction system using the unradioactive digoxigenin-labeled probe, and the specificity and sensitivity of probe were verified by blot hybridization method. Paraffin slice for in situ PCR and in situ hybridization was made and the location and distribution of ASPV RNA were detected in paraffin slices using in situ reverse transcription polymerase chain reaction, and the important factors which influenced the experiment results were optimized. ASPV mainly distributed in palisade tissue of mesophyll cells, external cortex of the tip, and the corresponding newborn vascular bundles. 20 min was the suitable digestive time for proteinase K. For the better amplication, RT reaction system should be above 0.2 U μL^-1 and 0.4 mmol L^-1 for RNasin and dNTPs respectively, 0.1-1.3 U μ L^-1 SuperScript Ⅱ, 0.6- 0.8 μmol L^-1 primer concentration, and above 0.5 U 100 μL^-1 LA Taq DNA polymerase. The suitable annealing temperature in PCR reaction was 60℃ with 35 cycles. The apical meristem of 0.25 mm was the region of virus-free.展开更多
In this study, in situ hybridization （ISH） was developed to detect avian influenza＇virus （AIV） in Madin-Darby canine kidney （MDCK） cells and formalin-fixed, paraffin-embedded chicken tissues. A cDNA probe corre...In this study, in situ hybridization （ISH） was developed to detect avian influenza＇virus （AIV） in Madin-Darby canine kidney （MDCK） cells and formalin-fixed, paraffin-embedded chicken tissues. A cDNA probe corresponding to a region of AIV nucleoprotein （NP） gene was synthesized and labeled with digoxigenin. Probe specificity was determined by AIV infected MDCK cells in vitro and the results showed that strong cytoplasmic staining was only detected in AIV-infected cells. Various tissues were collected from 12 h to 35 days post-infection （PI） following inoculation with the H9N2 subtype A1V. AIV was localized in the epithelial cells of the duodenum and cartilage of the throat and trachea at 12 h PI. Tissues from uninfected chickens were negative. The finding of this study indicated ISH was a sensitive and specific technique to detect and localize AIV as well as to study AIV pathogenesis.展开更多
文摘Dot-blot hybridization was applied in this paper to detect five viruses infecting cucurbitaceous crops, Zuccini yellow mosaic virus （ZYMV）, Watermelon mosaic virus （WMV）, Cucumber mosaic virus （CMV）, Papaya ringspot viruswatermelon strain （PRSV-W） and Squash mosaic virus （SqMV）, as a good alternative assay in seed health test and epidemiological and transgenic research. Digoxigenin-labelled cDNA probes of the five viruses were synthesized by PCR with the specific primers and applied in dot-blot hybridization to detect five viruses in crude extraction of the infected leaves. And three SqMV probes of different lengths （0.55, 1.6, and 2.7 kb, respectively） were designed to investigate the effect of hybridization. The results showed that the sensitivity for detecting the crude extraction of infected leaves by ZYMV, WMV, CMV, PRSV-W, and SqMV was down to 1：160, 1：160, 1：320, 1：160, and 1：320, respectively. Three SqMV probes of different length showed no differences on the sensitivity and specificity. The digoxigenin-labelled probes prepared by PCR could be used for accurate and rapid identification of 5 viruses infecting cucurbitaceous crops with good stabilities, sensitivities, specificity, and reproducibilifies.
基金supported by the National Natural Science Foundation of China (30360066)the National Key Technologies R&D Program of China (2003BA546C)the Foundation Science and Technology Commission Xinjiang Production and Construction Corps,China(NKB02SDXNK01SW)
文摘To understand the distribution of Apple stem pitting virus （ASPV） in tissues of pear tree and provide directly theorical and technical support for shoot tips detoxication, leaves and shoot tips of Korla pear were used as the materials, cDNA probe for ASPV was synthesized through RT-PCR reaction system using the unradioactive digoxigenin-labeled probe, and the specificity and sensitivity of probe were verified by blot hybridization method. Paraffin slice for in situ PCR and in situ hybridization was made and the location and distribution of ASPV RNA were detected in paraffin slices using in situ reverse transcription polymerase chain reaction, and the important factors which influenced the experiment results were optimized. ASPV mainly distributed in palisade tissue of mesophyll cells, external cortex of the tip, and the corresponding newborn vascular bundles. 20 min was the suitable digestive time for proteinase K. For the better amplication, RT reaction system should be above 0.2 U μL^-1 and 0.4 mmol L^-1 for RNasin and dNTPs respectively, 0.1-1.3 U μ L^-1 SuperScript Ⅱ, 0.6- 0.8 μmol L^-1 primer concentration, and above 0.5 U 100 μL^-1 LA Taq DNA polymerase. The suitable annealing temperature in PCR reaction was 60℃ with 35 cycles. The apical meristem of 0.25 mm was the region of virus-free.
文摘In this study, in situ hybridization （ISH） was developed to detect avian influenza＇virus （AIV） in Madin-Darby canine kidney （MDCK） cells and formalin-fixed, paraffin-embedded chicken tissues. A cDNA probe corresponding to a region of AIV nucleoprotein （NP） gene was synthesized and labeled with digoxigenin. Probe specificity was determined by AIV infected MDCK cells in vitro and the results showed that strong cytoplasmic staining was only detected in AIV-infected cells. Various tissues were collected from 12 h to 35 days post-infection （PI） following inoculation with the H9N2 subtype A1V. AIV was localized in the epithelial cells of the duodenum and cartilage of the throat and trachea at 12 h PI. Tissues from uninfected chickens were negative. The finding of this study indicated ISH was a sensitive and specific technique to detect and localize AIV as well as to study AIV pathogenesis.
